What Are Unique Molecular Identifiers

In the ever-evolving realm of genomic research, the amalgamation of cutting-edge technologies has catalyzed a paradigm shift, illuminating hitherto unexplored facets of the genome. Amidst this array of innovations, Unique Molecular Identifiers (UMIs) have ascended as formidable instruments, augmenting the precision and sensitivity of next-generation sequencing (NGS) endeavors. Within the confines of this exhaustive discourse, we embark upon an exploration of UMIs, meticulously dissecting their definition, manifold applications, and the seminal influence they have exerted on the landscape of genomic inquiries.

Understanding Unique Molecular Identifiers 

UMIs represent concise, randomly generated nucleotide sequences seamlessly integrated into individual DNA or RNA molecules during the preparatory phase of NGS library construction. These distinctive molecular tags serve dual functions: error rectification and molecular deduplication. Through the allocation of a unique UMI to each molecule preceding the amplification stage, investigators gain the capability to discern with precision between authentic biological signals and spurious artifacts stemming from sequencing methodologies.

Benefits of Unique Molecular Identifiers

Amplified Error Mitigation

One of the cardinal merits of UMIs is their pronounced potential to augment error rectification in NGS investigations. The judicious inclusion of UMIs allows researchers to efficaciously discern genuine sequencing variants from anomalies that are the byproducts of Polymerase Chain Reaction (PCR), sequencing, and base calling procedures. This upgrade in data accuracy markedly attenuates false-positive rates, thereby fostering the detection of rare alleles with formidable sensitivity.

Enhanced Molecular De-duplication

Owing to the integral nature of PCR in amplifying DNA fragments, there is an inevitable propensity for duplicate production, giving rise to the complication in the precise quantification of molecular diversity. UMIs tentatively tackle this issue by endowing each DNA fragment with a unique label, thereby enabling a clear distinction between PCR duplicates and legitimate molecular duplicates. This unique ability for molecular de-duplication ensures meticulous quantification of genomic regions of interest, thus anchoring a robust foundation for profound downstream analyses.

Unambiguous Determination of Molecular Diversity

UMIs also play a pivotal role in enabling the exact quantification of molecular diversity within a specimen. The strategy of designating each molecule with a specific identifier provides researchers with the ability to accurately gauge the prevalence of disparate species or variants in an intricately mixed sample. This proficiency becomes crucial in single-cell genomics, where analyzing heterogeneous cell populations necessitates accurate quantification of gene expression at the single-cell granularity.

Improved Accuracy and Sensitivity

Another cardinal advantage of UMIs stems from their aptitude to upgrade the accuracy and the sensitivity of NGS assays. Their innate ability to tag distinctive molecules with unique identifiers helps researchers in discerning PCR duplicates from authentic biological signals. This action considerably alleviates the effect of amplification biases and sequencing inaccuracies. This spike in accuracy becomes particularly critical in contexts where the detection of rare variants or the spotting of low-abundance transcripts is paramount.

How Do Unique Molecular Identifiers Work

The integration of UMIs into NGS experiments follows a systematic workflow encompassing several critical stages:

Library Preparation: UMIs are incorporated at the outset of library preparation. This initial phase involves the fragmentation of DNA or RNA molecules, followed by their tagging with adapter sequences.

Amplification: Post-library preparation, the tagged molecules undergo Polymerase Chain Reaction (PCR) amplification to achieve the requisite material for sequencing.

Sequencing: Subsequent to amplification, sequencing ensues, wherein each molecule undergoes multiple reads. Here, the UMI serves as a distinct marker facilitating error correction and de-duplication processes.

Data Analysis: The acquired sequencing data undergoes meticulous scrutiny employing bioinformatics tools. This phase involves the identification and elimination of PCR duplicates and sequencing errors, leveraging UMI data for enhanced accuracy.

In summary, the integration of UMIs within NGS experiments is a methodical process that spans from the inception of library preparation to the final stages of data analysis. Through each step, the UMI plays a pivotal role in ensuring precision and reliability in genomic sequencing endeavors.

Use of UMIs.Use of UMIs. (Neha Chaudhary et al, 2018)

Applications of UMIs

In the realm of genomics, the emergence of UMIs has sparked a paradigm shift in how researchers tackle diverse facets of genomic analysis. These brief, randomly generated nucleotide sequences have permeated various research domains, presenting unparalleled accuracy, sensitivity, and resolution. UMIs exhibit versatile utility across an array of sequencing methodologies where precise quantification or detection of rare mutations is imperative, or when input material is limited, including RNA-seq, single-cell RNA-seq and immune repertoire sequencing.

Single-Cell Genomics

Elucidating Cell-to-Cell Variability

The advent of single-cell genomics presents an efficient approach for scrutinizing the intrinsic heterogeneity tangled within multifaceted biological structures, such as tissues and tumours. UMIs play a central role in single-cell RNA sequencing (scRNA-seq) experiments, enabling precise quantification of gene expression metrics at the individual cellular level. By assigning each mRNA molecule a unique identifier, scientists are able to accurately pinpoint variations in gene expression profiles across cells, identifying rare cellular population subsets and principal curators of cellular identity and activity.

Profiling Singular Cell Types

UMIs are an indispensable tool for the profiling of singular cell types within heterogeneous clusters, providing unprecedented sensitivity. As each transcript is associated with a distinct identifier, scientists can confidently detect transcripts of low-abundance and rare cellular groupings, potentially harbouring essential insights into disease origin, immune response mechanisms, and critical developmental processes. This functionality offers immense value when studying rare cell populations such as circulating tumour cells, stem cells, and immune cell subsets, where conventional bulk sequencing methods may inadvertently mask pertinent biological signals.

Detecting Rare Genetic Modifications

Identifying rare genetic modifications poses serious difficulties in genomic research, particularly when it pertains to disease diagnosis, prognosis, and subsequent treatments. UMIs offer an innovative solution to these complications, significantly enhancing the sensitivity and accuracy of variant detection algorithms. By reducing the impact of polymerase chain reaction duplicates and sequencing errors, UMIs allow for the confident identification of low-frequency genetic alterations, somatic variants, and structural modifications within intricate genomic architectures. This functionality holds major implications for initiatives in precision medicine, where exact detection of rare genetic changes is integral to tailoring personalized treatment plans and promoting improved patient outcomes.

Quantifying Gene Expression Dynamics

UMIs further allow for detailed quantification of gene expression dynamics overtime, providing a window into the regulatory networks guiding essential biological procedures such as differentiation, development, and environmental responsive behaviours. By monitoring individual mRNA molecule abundance across various temporal checkpoints, researchers can illuminate the temporal facets of gene expression, isolate key transcriptional controllers, and disclose novel regulatory methodologies underpinning complex biological phenomena. The ability to analyze gene expression with such temporal precision is especially advantageous in longitudinal studies, where gene expression pattern shifts over a given period are indispensable for comprehensive understanding of disease progression, response to treatment, and therapeutic resistance.

At CD Genomics, we are dedicated to leveraging the potential of UMIs to propel genomic research forward and foster scientific progress. Through our specialized knowledge and state-of-the-art technologies, we empower researchers to untangle the intricacies of the genome and expedite the journey towards precision medicine.

Digital RNA Sequencing (UMI-RNA-Seq)
Single-Cell Sequencing
Single-cell RNA sequencing
mRNA Sequencing Service
Transcriptomics sequencing

Distinguishing Cell Barcode from UMI

Cell Barcoding: Tracing Cellular Lineage

Cell barcodes, short distinctive DNA sequences, act as unique identifiers in single-cell genomic research, tracing the cellular origin of sequencing reads. This assigns unique recognition to each cell within a heterogeneous pool, thereby permitting the accurate delineation of individual cellular gene expression profiles. With the leverage of cell barcoding, researchers can delve into cellular heterogeneity, thus illuminating the intricate dynamics governing biological systems with an unmatched degree of precision.

UMIs: Pioneering Error Correction

Meanwhile, UMIs perform a differential yet equally critical role in (NGS experiments. These distinctive sequences entwine with DNA fragments, thereby enabling an efficacious correction of errors and molecular de-duplication. While cell barcodes serve to pinpoint the cellular origin of sequencing reads, UMIs facilitate the rectification of PCR duplicates and sequencing inconsistencies, resulting in the enhancement of the fidelity of subsequent data interpretation. By marking each DNA fragment uniquely, UMIs provide researchers with an unprecedented level of confidence in the detection of rare alleles and quantification of gene expression levels.

UMIs in Single-Cell Genomics: A Quantum Leap in Genomic Research

Incorporating UMIs into genomic assays has resulted in a substantial increase in the sensitivity and specificity of these tests. Revolutionary platforms such as 10x Genomics make use of UMIs for rigorous error management and precise quantification of gene expression levels in scRNA-seq experiments. This integration of UMIs into library preparation protocols endows researchers with a forward leap in the exploration of cellular heterogeneity and contributes to an unraveling of intricate biological processes with unmatched resolution.

UMIs have spearheaded a revolution in single-cell genomics, as they allow for a precisely quantitative analysis of gene expression levels at the single-cell stratum. Notably, high-throughput platforms such as the Chromium system from 10x Genomics employ UMIs to tag discrete mRNA molecules. This allows researchers to deeply investigate transcriptional profiles among heterogeneous cell populations and characterize rare cell clusters. This detailed appraisal of cell-specific gene expression imprints creates a pathway towards identifying novel biomarkers and therapeutic targets.

Consequently, UMIs act as invaluable tools in RNA sequencing, driving our understanding of complex biological processes to unprecedented depths.

Exploring UMIs in RNA-Seq

UMIs represent a formidable stratagem in bolstering the precision, sensitivity, and reproducibility of RNA sequencing studies. By incorporating UMIs into protocols for library preparation, scientists are offered the capacity to surmount technical obstacles, specifically those associated with PCRduplicates and sequencing errors. This fosters an exact quantification of gene expression, all while enabling the detection of seldom transcripts. As RNA-Seq continually progresses in its indispensability in the realm of genomics, UMIs will persist in being integral in the investigation of gene regulation complexities and advancing our comprehension of biological systems.

Error Correction and Quantification

UMIs in RNA-Seq primarily exist to facilitate error rectification. During the amplification and sequencing phases of library preparation, incidences of PCR duplication and sequencing errors may impose inaccuracies into the measurement of gene expression. UMIs present scientists with the provision to recognize and eliminate these inaccuracies, culminating in a more refined quantification of RNA molecules and an enhanced reliability of subsequent analyses.

Applications of UMIs in RNA-Seq

Elucidating Gene Expression Dynamics UMIs are vital for the delineation of gene expression dynamics traversing various biological contexts. These elements permit the high-precision quantification of transcript profusion at the single-molecule scope, enabling researchers to decipher intricate regulatory networks. In addition, they can identify genes displaying differential expression and clarify underlying molecular mechanisms ruling biological processes – from development and disease progress to responses to environmental influences.

Identifying Rare Transcripts

The sensitivity granted by UMIs allows for the detection of rare transcripts, which are typically present at low abundance within a varied RNA assortment. This is particularly applicable in investigations focusing on infrequent cell populations, where traditional RNA-Seq techniques may fail to identify fleeting or rare gene expression events. With the aid of UMIs, it becomes feasible to discover new biomarkers, categorize distinct cell subsets, and gain insight into cell heterogeneity with unparalleled resolution.

Quantifying Allelic Expression

UMIs also find utility in the quantification of allelic expression, permitting differentiation amongst transcripts arising from distinct alleles housed within diploid genomes. Such a feature proves to be crucial in researching allele-specific gene expression, genetic imprinting, and allele-specific regulatory mechanisms. With accurate quantification of allelic expression patterns, it's possible to illuminate the genetic foundation of complex traits and diseases, laying a solid basis for personalized medicine and novel targeted therapeutic strategies.

Challenges and Considerations

While UMIs endow numerous advantages in RNA-Seq experiments, the effective usage of these elements necessitates a thoughtfully planned experimental layout and robust data breakdown structures. Investigators must take into account factors relating to UMI length, sequencing depth, and bioinformatics tools necessary for error rectification and quantification. Furthermore, it is of paramount importance that UMIs are correctly handled during data analysis to ensure precise interpretation of results, and to minimize potential biases introduced during the library preparation and subsequential sequencing phases.


To conclude, the advent and implementation of UMIs signify a revolutionary transformation in genomic studies, providing an unrivaled edge in refining error correction and achieving precise molecular quantification. The integration of UMIs into NGS experimentation enables the scientific community to delve into the complexities of genomes and transcriptomes with a hitherto unseen level of granularity. Positioned at the nexus of technological advancements, CD Genomics remains steadfast in its commitment to catalyzing strides in genomic fields through pioneering technologies and state-of-the-art research platforms. By harnessing the potential of UMIs, CD Genomics not only facilitates unraveling the intricacies of genomes but also contributes towards unveiling novel insights into aspects of human health and disease.


What is a UMIs in NGS

UMIs, or Unique Molecular Identifiers, have fundamentally transformed the domain of NGS by innovating how researchers examine genomic information. As distinctive, succinct sequence tags incorporated into each DNA fragment before amplification, UMIs enable intricate and reliable molecular tracking throughout the sequencing procedure. This signifies a considerable departure from established methods, presenting scientists with novel and unparalleled insights into molecular heterogeneity. With UMIs, researchers now possess a significantly more precise framework for identifying and quantifying genetic variants – a pivotal development in the realm of genomic studies.

How do unique molecular identifiers work?

UMIs operate by allocating a unique nucleotide sequence to every DNA or RNA molecule individually at the library preparation phase of NGS. These brief, randomly generated sequences function as molecular markers, distinctively marking each molecule within the specimen. Throughout subsequent amplification and sequencing stages, UMIs persist linked to their corresponding molecules, empowering researchers to monitor and differentiate among them. Through the utilization of UMIs, investigators can proficiently discern and rectify errors arising from PCR, sequencing, and base calling procedures, thus amplifying the precision and sensitivity of NGS endeavors.

What Are UMIs used for?

UMIs are instrumental in numerous facets of genomic research, chiefly dedicated to refining the precision and sensitivity of NGS investigations. These distinctive molecular markers are harnessed for error rectification, molecular deduplication, and the assessment of molecular heterogeneity within samples. By meticulously discerning genuine biological signals from artifacts introduced during sequencing procedures, UMIs empower researchers to identify rare alleles with unparalleled sensitivity. Furthermore, UMIs facilitate the meticulous quantification of gene expression levels at the single-cell resolution, particularly pivotal in single-cell genomics where the analysis of diverse cell populations mandates precise quantification.

What is the difference between cell barcode and UMI?

While both cell barcodes and UMIs act as molecular identifiers in NGS experiments, they fulfill distinct roles. A cell barcode, also referred to as a cell index or sample index, comprises a concise DNA sequence utilized to differentiate between various biological samples or cells. It denotes the origin of the sequencing read, signifying the cell from which the RNA or DNA molecule was derived. Conversely, a UMI represents a short, randomly generated nucleotide sequence that functions to tag individual DNA or RNA molecules within a sample uniquely. UMIs contribute to error correction and molecular deduplication by precisely labeling each molecule, enabling researchers to discriminate genuine biological signals from artifacts introduced during sequencing processes. Consequently, while both are indispensable for precise genomic analysis, cell barcodes identify the provenance of sequencing reads, whereas UMIs facilitate error correction and quantification at the molecular level.


  1. Kou R, Lam H, Duan H, Ye L, Jongkam N, Chen W, Zhang S, Li S. Benefits and Challenges with Applying Unique Molecular Identifiers in Next Generation Sequencing to Detect Low Frequency Mutations. PLoS One. 2016
  2. Bieler, J., Kubik, S., Macheret, M. et al. Benefits of applying molecular barcoding systems are not uniform across different genomic applications. J Transl Med 21, 305 (2023).
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